Chung Khoa, Booth Michael J
Department of Chemistry, University of Oxford Mansfield Road OX1 3TA Oxford UK
Department of Chemistry, University College London 20 Gordon Street London WC1H 0AJ UK.
Chem Sci. 2023 Oct 13;14(44):12693-12706. doi: 10.1039/d3sc02761a. eCollection 2023 Nov 15.
Plasmids are ubiquitous in biology, where they are used to study gene-function relationships and intricate molecular networks, and hold potential as therapeutic devices. Developing methods to control their function will advance their application in research and may also expedite their translation to clinical settings. Light is an attractive stimulus to conditionally regulate plasmid expression as it is non-invasive, and its properties such as wavelength, intensity, and duration can be adjusted to minimise cellular toxicity and increase penetration. Herein, we have developed a method to site-specifically introduce photocages into plasmids, by resynthesising one strand in a manner similar to Kunkel mutagenesis. Unlike alternative approaches to chemically modify plasmids, this method is sequence-independent at the site of modification and uses commercially available phosphoramidites. To generate our light-activated (LA) plasmids, photocleavable biotinylated nucleobases were introduced at specific sites across the T7 and CMV promoters on plasmids and bound to streptavidin to sterically block access. These LA-plasmids were then successfully used to control expression in both cell-free systems (T7 promoter) and mammalian cells (CMV promoter). These light-activated plasmids might be used to remotely control cellular activity and reduce off-target toxicity for future medical use. Our simple approach to plasmid modification might also be used to introduce novel chemical moieties for advanced function.
质粒在生物学中普遍存在，可用于研究基因功能关系和复杂的分子网络，并具有作为治疗工具的潜力。开发控制其功能的方法将推动其在研究中的应用，也可能加速其向临床应用的转化。光作为一种有吸引力的刺激因素，可用于条件性调节质粒表达，因为它是非侵入性的，其波长、强度和持续时间等特性可以调节，以将细胞毒性降至最低并增加穿透力。在此，我们开发了一种方法，通过以类似于Kunkel诱变的方式重新合成一条链，将光笼位点特异性地引入质粒中。与化学修饰质粒的其他方法不同，该方法在修饰位点不依赖于序列，并且使用市售的亚磷酰胺。为了生成我们的光激活（LA）质粒，在质粒上T7和CMV启动子的特定位点引入了可光裂解的生物素化核碱基，并与链霉亲和素结合以空间位阻方式阻止访问。然后，这些LA质粒成功用于在无细胞系统（T7启动子）和哺乳动物细胞（CMV启动子）中控制表达。这些光激活质粒可用于远程控制细胞活性并降低未来医学应用中的脱靶毒性。我们简单的质粒修饰方法也可用于引入具有先进功能的新型化学基团。
